The subject of the invention is the provision of stable salts of hyperforin and adhyperforin which are capable of pharmacological activity as they are or by release of the hyperforin or adhyperforin. A fundamental aspect of the invention relates to the provision of a method of enriching or purifying hyperforin and adhyperforin from extracts of St. John""s wort by means of precipitation in the form of these stable salts. Another especially important subject of the invention comprises the provision of new active substances for controlling Alzheimer""s disease (hereinafter abbreviated to xe2x80x9cADxe2x80x9d) by treating the cause of the disease.
A further important aspect of the present invention relates to the provision of active-substance combinations which can be used to treat the cause of AD, and at the same time eliminate, considerably improve, or at least halt the progression of psychopathological concomitant phenomena which frequently arise in association with AD, such as anxiety, depressive illnesses, and cognitive disturbances.
The amyloid peptide Axcex21-42, a processed product of Alzheimer Precursor Protein APP, plays a central role in the occurrence of AD [Lamb, B. T.: Presenilins, amyloid-xcex2 and Alzheimer""s Disease. Nature Med. 3 (1997) 28-29. Selkoe, D. J.: Alzheimer""s Disease: Genotypes, Pheno-type, and Treatments. Science 275 (1997) 630-631]. This hypothesis is supported by the following experimental findings:
APP Missense mutations (patients with familial AD) lead to an increased release of Axcex21-42 [Scheuner, D. et al.: Secreted amyloid xcex2-protein similar to that in the senile plaques of Alzheimer""s disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer""s disease. Nature Med. 2 (1996) 864-870].
Mutations in presenilin 1 and presenilin 2 (patients with familial AD) similarly lead to an increase in released Axcex21-42 [Scheuner, D. et al]. Transgenic mice, which overexpress mutated APP, develop age-dependent deposits of Axcex2 and show cognitive disturbances [Games, D. et al.: Alzheimer-type neuropathology in transgenic mice overexpressing V717F xcex2-amyloid precursor protein. Nature 373 (1995) 523-527. Hsiao, K. et al.: Correlative memory deficits, Axcex2 Elevation, and Amyloid Plaques in Transgenic Mice. Science 274 (1996) 99-102].
The proteolytic cleavage of the pathogenic Axcex2 from the Alzheimer Precursor Protein APP is mediated by xcex2- and xcex3-secretase, the molecular identity of which is unknown. xcex1-Secretase processes APP to a soluble form (sAPP) and a cytoplasmatic residue. The cleavage of xcex1-secretase lies within Axcex2, with the result that in this case no pathogenic Axcex2 arises. The molecular identity of xcex1-secretase is similarly unknown.
xcex1-Secretase is stimulated by acetylcholine, mediated by the muscarinic receptors m1 and m3 [Nitsch, R. M. et al.: Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science 258 (1992) 304-307]. The cellular mediator is protein kinase C (xe2x80x9cPKCxe2x80x9d). This is also confirmed by experiments which, following direct stimulation of PKC by phorbol ester, reach the same result [Buxbaum, J. D. et al.: Processing of Alzheimer beta/A4 amyloid precursor protein: Modulation by agents that regulate protein phosphorylation. Proc. Natl. Acad. Sci. USA 87 (1990) 6003-6006].
Tacrine, the most successful therapeutic agent to date against AD is an acetylcholine inhibitor [Giacobini, E.: Cholinomimetic therapy of Alzheimer disease: Does it slow down deterioration? In Recent Advances in the Treatment of Neurodegenerative Disorders and Cognitive Dysfunction, Int. Acad. Biomed. Drug Res. 7 (1994) 51-57. Racagni, G. et al., eds. Basel: Karger].
This may be interpreted as indirect stimulation of xcex1-secretase by the following signal chain: Tacrine inhibits acetylcholinesterase. The concentration of acetylcholine is thereby increased. Acetylcholine activates the PKC via the muscarinic receptors m1 and m3. By this means the activity of xcex1-secretase is increased. In consequence the quantity of pathogenic Axcex2 is lowered.
From these findings it can be concluded that selective activation of PKC can be an effective therapeutic starting point to inhibiting the production of amyloidogenic Axcex2 and thus to the treatment of AD. Since, of all 11 PKC isoenzymes, the xcex3-form is the only sub-type to be expressed exclusively in neuronal cells, substances which stimulate PKC-xcex3 represent a new starting point to the therapy of AD. Moreover, all substances or processes which stimulate xcex1-secretase or inhibit xcex2- and xcex3-secretase are suitable for preventing the release of pathogenic Axcex2 and thus for treating the cause of AD.
The phloroglucin derivative hyperforin is one of the principal ingredients in fresh St. John""s wort. It is associated with its homologue adhyperforin in a lower concentration. As both substances are highly unstable to light and the influence of air, their content declines even when the fresh plant is dried. By fast and careful drying followed by suitable extraction methods, extracts with a content of about 3-60% hyperforin/adhyperforin can be obtained [DE 19619512 C1]. 
However, without addition of appropriate stabilisers, hyperforin is not stable, and can therefore be obtained and stored in an enriched or pure form only by use of expensive techniques.
Reference has already been made to the importance of hyperforin for achieving the antidepressant efficacy of St. John""s wort extracts in EP-A-0599307. Since then it has been scientifically proven that, on the basis of its pharmacological profile, hyperforin exerts a considerable influence in the medical treatment of depression and other serotonin-dependent diseases [S. S. Chatterjee et al., hyperforin and hypericum extract, Interactions with some Neurotransmitter Systems (SL-82), 2nd Intern. Congress on Phytomedicine, Sep. 11-14, 1996, Munich. See also: Pharmacopsychiatry 1998, 31 Suppl. I, 1-60].
Alzheimer""s dementia (AD) is a serious disease of gradual onset which affects a considerable proportion of the population especially the elderly. It is characterised by initial forgetfulness, then increasing memory disturbances and losses of other cognitive abilities. It concludes with complete mental degeneration and loss of personality, and takes an ultimately fatal course. To date, no satisfactory, cause-orientated therapy for AD is available [K. Mendla, Die Alzheimer-Krankheit: Neue Ansxc3xa4tze in der Pharmakotherapie (1996). Pharm.Ztg. 141, 351-356].
The technical problem underlying the invention thus consists in the fact that, firstly, there is no known technically satisfactory method for obtaining and stabilising pure or greatly enriched hyperforin and adhyperforin, severely impeding the isolation, storage and use of these substances; secondly, there is a deficiency of active substances for the cause-orientated therapy of Alzheimer""s disease, resulting in massive financial outlays within the social services. The problem of the invention is to help eliminate these defects.
This problem is resolved according to the invention by
the new salts of hyperforin and adhyperforin according to patent claims 1 to 5;
the method of manufacturing these salts according to Claim 6;
the method of enriching or purifying hyperforin and adhyperforin in the form of these salts according to Claims 7 and 8;
the use of these salts for maintaining stable stocks of hyperforin, adhyperforin and their mixtures according to Claim 9;
the pharmaceutical preparation according to Claim 10, and
the new use of hyperforin, adhyperforin and their mixtures as medicinal products for the treatment of AD (2nd medical indication).
It was surprisingly found that the instability of hyperforin or of adhyperforin can be completely eliminated or at least considerably reduced by conversion of the substance into suitable salts of general formula I
[Axe2x88x92]m[B]p+xe2x80x83xe2x80x83(I)
No salts of hyperforin are known to date.
In formula I, m is a whole number from 1 to 3 and [Axe2x88x92] is the anion of hyperforin or adhyperforin, where n=0 or 1 (general formula II): 
and [B]p+ is either
an ion of an alkali metal, preferably Li+, Na+ or K+, where p=1, or an ammonium ion of a salt-forming nitrogen base of general formula III 
xe2x80x83wherein the residues R1, R2 and R3,
independently of one another, are a hydrogen atom, a straight-chain or branched alkyl, cycloalkyl, bicycloalkyl, tricycloalkyl, alkenyl, alkynyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl group, or a derivative of the said groups substituted with one or more hydroxy, alkoxy, aryloxy, alkanoyl, aroyl, carboxy, alkoxycarbonyl, amino, alkylamino, hydroxylamino, amido, carbamoyl, ureido, amidino, guanidino, cyano, azido, mercapto, alkylthio, alkylsulphoxy, alkylsulphonyl, alkylsulphenyl, aminosulphonyl, fluoro, chloro, bromo, iodo, alkyl or perfluoroalkyl residue(s),
xe2x80x83or wherein the residues R1 and R2
together with the N atom are an azetidine, pyrrolidine, pyrroline, piperidine, piperazine, homopiperazine, morpholine, thiomorpholine, pyridine, di- or tetrahydropyridine, pyrimidine, pyrazine, azepine, dihydroazepine, oxazepine, diazepine, imidazole, pyrazole, oxazole, or thiazole ring, or one of the said rings which exhibits aliphatic, heteroaliphatic, aromatic or heteroaromatic rings condensed on to it and/or is substituted with one or more hydroxy, alkoxy, aryloxy, alkanoyl, aroyl, carboxy, alkoxycarbonyl, amino, alkylamino, hydroxylamino, amido, carbamoyl, ureido, amidino, guanidino, cyano, azido, mercapto, alkylthio, alkylsulphoxy, alkysulphonyl, alkylsulphenyl, aminosuiphonyl, fluoro, chloro, bromo, iodo, alkyl or perfluoroalkyl residue(s),
xe2x80x83and wherein the residue R4
is a hydrogen atom or a straight-chain or branched alkyl group, in which p=m and gives the total number of positive charges of the residue [B].
The N,N-dicyclohexylamine salts of hyperforin and adhyperforin and their mixtures are especially preferable.
From the above definition of the base B serving as a salt-former it emerges that a large number of basic compounds of nitrogen are suitable for satisfactorily increasing the stability of hyperforin or adhyperforin, both of which are unstable in the uncharged form.
Suitable bases are e.g.:
aliphatic and cycloaliphatic amines or polyamines such as mono-, di-, or tri-(C3 to C20)alkylamines, aminoethanol, methylaminoethanol, dimethylaminoethanol, choline, 2-hydroxy-1,1-dimethylethylamine, tris(hydroxymethyl)methylamine, N-methyl-D-glucamine, ethylenediamine, dicylohexylamine, N-cyclohexyl-N-3-aminopropylamine, 1-aminoadamantane or 1-amino-3,5-dimethyladamantane, optionally substituted with one or more hydroxyl groups,
cyclic or heterocyclic amines such as pyrrolidine, piperidine, morpholine, piperazine, N-methylpiperazine or N-methylhomopiperazine, optionally substituted with one or more lower alkyl- (=C1 to C4-alkyl) or hydroxyl residues,
possibly substituted aromatic, heteroaromatic, arylaliphatic or heteroarylaliphatic amines such as benzylamine, 3,4,5-trimethoxybenzylamine, veratrylamine, phenethylamine, homoveratrylamine, N-methylhomoveratrylamine, 4-aminopyridine, tacrine and analogues, imipramine, desipramine, selegiline, nicotine, pindolol,
amino acid esters and amides such as methyl, ethyl, propyl or isopropyl esters and amides of glycine, alanine, phenylalanine, leucine, isoleucine, methionine, proline, valine, sarcosine, pipecolic acid,
as well as basic amino acids such as lysine or arginine or their amides.
Bases which are especially suitable are those that are themselves active substances and have the same medical indications as hyperforin, or support its therapeutic use. Those coming into consideration are primarily basic active substances with the indications:
Alzheimer""s Disease (AD), e.g.
Acetylcholinesterase inhibitors (e.g. amiridine, donezepil, ensaculine, eptastigmine, galanthamine, huperzine A, 7-methoxytacrine, physostigmine, SDZ-ENA-713 (Exelon), SM-10888, suronacrine, tacrine, velnacrine), cholinergic activators, NMDA antagonists (e.g. memantine), glutamine-receptor antagonists, serotonin agonists and antagonists (e.g. adatanserin), monoamine oxidase inhibitors (e.g. tranylcypromine, selegelin), PKC activators and xcex1-secretase inhibitors, tyrosine kinase antagonists, muscarinic agonists (e.g. arecoline, BIBN 99, itameline, milameline, talsaclidine, xanomeline, YM796),
Antidepressants, e.g.
amitryptiline, dibenzepin, desipramine, desitryptiline, doselupine, doxepin, clomipramine, fluoxetine, fluvoxamine, imipramine, lofepramine, maprotiline, moclebemide, mianserin, nortriptyline, opipramol, paroxetine, tranylcypromine, trazodone, trimipramine, viloxazine,
Anxiolytics, e.g.
chlorprothixene, dixyrazine, fluphenazine, levomepromazine, melperone, perphenazine, promazine, promethazine, pritiphendyl, sulpiride, tandospirone, thioridazine, trifluoperazine, zuclopenthixol,
Calcium antagonists (with basic side chain) e.g.
amlodipine, azelnidipine, bamidipine, benidipine, cronidipine, edrecolomab (AE0047), efonidipine, elgodipine, lercanidipine, manidipine, nicardipine, palodipine, verapamil,
Dyspepsia therapeutic agents and prokinetics, e.g.
cisapride, metoclopramide, renzapride (5-HT4 agonists),
xcex2-receptor blockers, e.g.
atenolol, alprenolol, carazolol, propranolol, labetalol, mepindolol, metoprolol, oxprenolol, penbutolol, pindolol, bupranolol, bunitrolol, metipranolol, nadolol,
Nootropics, e.g.
lomerizine, nebracetam, pramiracetam, SNK-882.
Salts of hyperforin and adhyperforin with basic active substances of this type form an especially innovative partial aspect of the present invention, as they not only increase the stability of the therapeutically active hyperforin, but also, in consequence of their intrinsic therapeutic action, allow an especially appropriate combination of mutually potentiating or mutually supporting active principles.
The salts according to the invention can be manufactured in various ways. In the following explanation of the method, the term xe2x80x9chyperforinxe2x80x9d in all cases also means the homologue adhyperforin and mixtures of the two substances. xe2x80x9cLowerxe2x80x9d always means xe2x80x9cC1 to C4-xe2x80x9d.
Method A: Hyperforin is dissolved in a lower alkanol (e.g. methanol, ethanol, propanol or isopropanol), preferably under protective gas and with exclusion of light, reacted with the solution of an equimolar quantity of alkali-metal hydroxide or alkali-metal lower alkoxide (e.g. sodium methoxide or ethoxide) in one of the above-mentioned lower alkanols, the solution is evaporated, taken up with water and lyophilised. Stable, colourless to cream-coloured alkali salts of hyperforin are obtained in the form of a powder. Alternatively the hyperforin can also be dissolved directly in the solution of the alkali metal lower alkoxide and worked up as above.
Method B: Hyperforin is dissolved in an aprotic solvent selected from the group of apolar C1-C10 alkanes and C1-C10 cycloalkanes e.g. pentane, hexane, heptane, octane, isooctane, or cyclohexane, optionally with addition of small quantities of a lower alkanol (e.g. methanol, ethanol, or isopropanol), preferably under protective gas and with exclusion of light, this solution is reacted with the equimolar quantity of the basic component B or with a solution of the same in one of the above-mentioned solvents or in a lower halogenoalkane, e.g. dichloromethane or chloroform, or in a lower ether, e.g. diethyl ether, diisopropyl ether, tert.-butyl methyl ether or tetrahydrofuran, or in a lower ketone, e.g. acetone or methyl ethyl ketone, the mixture concentrated if necessary, the precipitating salt separated, if necessary recrystallised, and dried under vacuum. Crystalline or amorphous ammonium salts of hyperforin are obtained in the form of a powder.
If the base B contains more than one basic centre capable of salt formation, if desired correspondingly smaller quantities, e.g. xc2xd or ⅓-molar quantities of base B may be used, so that the hyperforin/base ratio is m/p.
Method C: Hyperforin is dissolved in a lower alkanol (e.g. methanol, ethanol, or isopropanol), preferably under protective gas and with exclusion of light, this solution is reacted with the equimolar quantity of the basic component B or with a solution of the same in one of the above-mentioned solvents, the mixture is evaporated, taken up in water, and lyophilised. Crystalline or amorphous ammonium salts of hyperforin are obtained in the form of a powder.
Method D: Hyperforin is dissolved in a lower alkanol (e.g. methanol, ethanol, or isopropanol), preferably under protective gas and with exclusion of light, this solution is reacted with an equimolar quantity of the basic component B in water, the lower alcohol is removed by distillation under vacuum, and the remaining aqueous mixture is lyophilised, if necessary after addition of water. The powdery salt obtained is if necessary re-crystallised from a lower alcohol, alcohol/water mixture or from a lower ester.
Method for the Enrichment or Pure Isolation of Hyperforin and Adhyperforin From St.-John""s Wort Extracts
To date the purification of hyperforin from St.-John""s wort extract has been possible only with the use of very expensive chromatographic methods which were furthermore made technically unacceptable by the high instability of hyperforin and its homologue to light and to the oxygen in air (P. Maisenbacher, Universitxc3xa4t Txc3xcbingen, Diss. 1991. R. Burgdxc3x6rfer, Universitxc3xa4t Marburg, Diss. 1987).
A surprisingly simple and cost-reducing solution to this technical problem has now been found which consists in dissolving St.-John""s wort extracts, e.g. a CO2 extract with a 20-80% hyperforin/adhyperforin content, in a suitable solvent selected from the series of apolar C1-C10 alkanes and cycloalkanes, e.g. pentane, hexane, heptane, octane, iso-octane, or cyclohexane, optionally with addition of small quantities of a lower alkanol (e.g. methanol, ethanol or isopropanol), preferably under protective gas and with exclusion of light, this solution is reacted with the at least equimolar quantity of the basic component B or a solution of the same in one of the above-mentioned solvents or in a lower halogenoalkane, e.g. dichloromethane or chloroform, or in a lower ether e.g. diethyl ether, diisopropyl ether, tert.-butylmethyl ether or tetrahydrofuran, or in a lower ketone, e.g. acetone or methyl ethyl ketone, the mixture is concentrated if necessary, the precipitating salt is separated, if necessary re-precipitated and/or re-crystallised and dried under vacuum. Crystalline or amorphous ammonium salts of hyperforin/adhyperforin or of a mixture of them are obtained in the form of a powder.
Salt-forming amines especially suitable for this method are cycloaliphatic (e.g. dicyclohexylamine) araliphatic (e.g. benzylamine and its methoxy-substituted derivatives), heterocyclic or heteroaromatic amines (e.g. 4-aminopyridine).
Hyperforin/adhyperforin can be readily obtained in a pure form from the crystalline and storage-stable salts by acidification, preferably with an organic acid (e.g. citric acid or tartaric acid), and subsequent distribution between one of the listed solvents and water, and either used as they are or transferred to other desired salts. To this end the hyperforin salt is dissolved or suspended in the selected solvent (e.g. methyl-tert.butyl ether or ethyl acetate), under a protective-gas atmosphere and exclusion of light, reacted with the at least equimolar quantity of the acid dissolved in water, stirred until completely dissolved, the aqueous phase is separated, and the organic phase is evaporated gently after washing with water.
The present invention further relates to medicinal products which, in addition to non-toxic, inert pharmaceutically suitable carrier substances, also contain one or more of the hyperforin and/or adhyperforin salts according to the invention, or which consist of one or more of the hyperforin and/or adhyperforin salts, as well as methods of manufacturing these medicinal products.
Non-toxic, inert pharmaceutically suitable carrier substances are to be understood as solid, semi-solid or liquid diluents, fillers and formulation adjuvants of all kinds.
Suitable solid or liquid galenical preparations of the medicinal products according to the invention are e.g. tablets, capsules, sugar-coated tablets, suppositories, syrups, emulsions, suspensions, drops or injectable solutions, as well as products with slow release of the active substance.
As carriers or diluents one should name e.g. various sugars or starches, cellulose derivatives, magnesium carbonate, gelatins, oils of animal or vegetable origin, polyethylene glycols, water or other physiologically safe solvents, as well as water-containing buffers, which may be rendered isotonic by addition of salts or glucose. Surfactant substances, colourings, flavourings, stabilisers and preservatives may also find a use as further additives in the medicinal products according to the invention.
The therapeutically effective compounds are present in the medicinal products listed above preferably in a concentration of about 0.5 to 95% of the overall mixture.
The medicinal products are manufactured using methods familiar to the person skilled in the art, e.g. by mixing of the active substance(s) with the carrier substances and additives and further processing to produce the desired galenical form.
The invention also relates to the use of the active substances of the invention, and of the medicinal products manufactured from them, in human medicine for the therapy or prophylaxis of Alzheimer""s disease.
Finally, the invention relates to the use of the substances hyperforin and/or adhyperforin, known ingredients of extracts used for therapeutic purposes, if necessary together with a pharmaceutically safe carrier or diluent, in human medicine for the therapy or prophylaxis of Alzheimer""s disease (2nd medical indication)
The active substances or medicinal products of the invention may be administered orally, parenterally, intravenously and/or rectally. In human medicine, the active substances are preferably administered in doses amounting in total to 0.01 to 10, in particular 0.05 to 5 mg/kg body weight per 24-h period, if necessary in the form of several unit doses. The total amount is administered in 1 to 5, preferably in 1 to 3 unit doses. The dosage and timing of the doses, and the choice of appropriate mode of administration can easily be accomplished by anyone skilled in the art on the basis of his or her scientific knowledge.